Next-generation mapping: a novel approach for detection of pathogenic structural variants with a potential utility in clinical diagnosis
Hayk Barseghyan, Wilson Tang, Richard T Wang, Miguel Almalvez, Eva Segura, Matthew S Bramble, Allen Lipson, Emilie D Douine, Hane Lee, Emmanuèle C Délot, Stanley F Nelson, Eric Vilain, Hayk Barseghyan, Wilson Tang, Richard T Wang, Miguel Almalvez, Eva Segura, Matthew S Bramble, Allen Lipson, Emilie D Douine, Hane Lee, Emmanuèle C Délot, Stanley F Nelson, Eric Vilain
Abstract
Background: Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient's phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100-300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations.
Methods: To overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene.
Results: We identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45-250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing.
Conclusions: We showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.
Keywords: Bionano; DMD; Duchenne muscular dystrophy; Nanochannel; Next-generation mapping; Optical mapping; Structural variants.
Conflict of interest statement
Ethics approval and consent to participateResearch involving human participants, human material, and human data have been performed in accordance with the principles of the Helsinki declaration and approved protocols by the UCLA Institutional Review Board: Genetic Modifiers of Duchenne and Becker Muscular Dystrophy (IRB#12-000769); Molecular Genetics of Degenerative Disease (IRB#11-001087). Informed consents were obtained during patients’ visits to the Pediatric Neuromuscular Clinic.
Consent for publicationParticipants have consented by approved IRB protocols to share their de-identified information.
Competing interestsThe authors declare that they have no competing interests.
Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Source: PubMed